The enzymic catalysis of chemical reactions is well-documented in the literature. It is well known to employ biocatalysts, such as microorganisms that contain enzymes, for conducting chemical reactions, or to use enzymes that are free of microorganisms. It is known that various ethylenically unsaturated monomers can be prepared by converting a substrate starting material into the desired monomer by use of a biocatalyst.
Nitrile hydratase enzymes are known to catalyse the hydration of nitrites to the corresponding amides. Typically nitrile hydratase enzymes can be synthesized by a variety of microorganisms, for instance microorganisms of the genus Bacillus, Bacteridium, Micrococcus, Brevibacterium, Corynebacterium, Pseudomonas, Acinetobacter, Xanthobacter, Streptomyces, Rhizobium, Klebsiella, Enterobacter, Erwinia, Aeromonas, Citrobacter, Achromobacter, Agrobacterium, Pseudonocardia, Rhodococcus and Comamonas. 
It is known to produce acrylamide from acrylonitrile using as a catalyst nitrile hydratase. When producing these products biologically it is desirable to employ an enzyme which is capable of producing aqueous solutions of acrylamide in high concentration and yet is not poisoned by acrylonitrile and high concentrations of acrylamide.
A review paper by Yamada and Kobayashi, Biosci. Biotech. Biochem 60: 1391-1400 (1996) charts the development of the biocatalysed process for the production of acrylamide monomer up to a concentration of 50%. This review describes the three generations of catalyst developed for the industrial production of acrylamide culminating with Rhodococcus rhodochrous J1, a bacterium that requires cobalt as part of the nitrile hydratase enzyme which catalyses the formation of acrylamide from acrylonitrile. The nitrile hydratase is synthesised in very high levels in the bacterium due to the presence of urea as an inducer in the culture medium.
A paper by Nawaz et al., Arch. Microbiol. 156:231-238 (1991), entitled ‘Metabolism of acrylonitrile by Klebsiella pneumoniae’ describes the isolation and growth of the bacterium K. pneumoniae and its subsequent rapid utilisation of acrylonitrile and formation of acrylamide which was then further hydrolysed to acrylic acid. The organism was isolated using an enrichment culture technique with acrylonitrile as the sole nitrogen source at pH 7.5.
Takashima et al., J. Indust. Microbiol. Biotechnol. (1998), Nitrile hydratase from a thermophilic Bacillus smithii, describes the characteristics of a thermophilic bacterium which synthesises nitrile hydratase. The nitrile hydratase has high acrylonitrile converting activity and the highest activity was at pH 10.5 or above. This would suggest for optimum activity to be achieved for this enzyme, the reaction solution would have to be buffered at this high pH.
Ramakrishna and Desai Biotechnol. Lett. 15: (3) 267-270 (1993) describes the superiority of cobalt induced acrylonitrile hydratase of Arthrobacter sp. IPCB-3 for conversion of acrylonitrile to acrylamide compared with an iron containing nitrile hydratase in this organism. This bacterium requires cobalt, and urea as a co-factor and inducer respectively to give the highest nitrile hydratase activity. Although the cobalt containing nitrile hydratase of this organism appears to have good acrylonitrile tolerance, at acrylamide concentrations of greater than 25% the enzyme activity was greatly reduced.
Various strains of the Rhodococcus rhodochrous species have been found to very effectively produce nitrile hydratase enzyme. EP-0 307 926 describes the culturing of Rhodococcus rhodochrous, specifically strain J1 in a culture medium that contains cobalt ions. The nitrile hydratase can be used to hydrate nitriles into amides, and in particular the conversion of 3-cyanopyridine to nicotinamide. This organism is further described in EP-0362829, which describes a method for cultivating bacteria of the species Rhodococcus rhodochrous comprising at least one of urea and cobalt ion for preparing the cells of Rhodococcus rhodochrous having nitrile hydratase activity. Specifically described is Rhodococcus rhodochrous J1.
Rhodococcus rhodochrous J1, is used commercially to manufacture acrylamide monomer from acrylonitrile and this process has been described by Nagasawa and Yamada, Pure Appl. Chem. 67: 1241-1256 (1995).
Leonova et al., Appl. Biochem. Biotechnol. 88: 231-241 (2000) entitled, “Nitrile Hydratase of Rhodococcus”, describes the growth and synthesis of nitrile hydratase in Rhodococcus rhodochrous M8. The nitrile hydratase of this strain is induced by the presence of urea in the medium, which is also used as a nitrogen source for growth by this organism. Cobalt is also required for high nitrile hydratase activity. This literature paper mainly looks at induction and metabolic effects.
Each of the aforementioned references describe bacteria that produce nitrile hydratase enzymes. All of these disclosures require that the bacteria are grown at approximately neutral pH.
The genus Dietzia was first described by Rainey et al., Int. J. Syst. Bacteriol 45: 32-36 (1995). Dietzia maris became the type species for the genus. In 1999 a further species addition was made: Dietzia natronolimnaea, this species first being described by Grant et al., Extremophiles 2: 359-366 (1998) in the publication entitled ‘Dietzia natronolimnaios sp. Nov., a new member of the genus Dietzia isolated from an East African soda lake’.
The Dietzia natronolimnalos strain isolated by these researchers 15LN1 (CBS 107.95) is an alkaliphile and as such grows at high pH (10) and in addition it grows in culture media containing high salt concentrations (40 g/l).
The Dietzia genus has been described for the catalysis of the synthesis of saturated compounds. For instance, WO-A-02/12530 describes a process for preparing 3-hydroxycarboxylic acid by the hydrolysis of 3-hydroxynitrile using Dietzia sp. ADL1 (ATCC PTA-1854).
A process for the preparation of glycine from glycinonitrile using microorgansims is described in WO01/048234. A number of microbial species are described in this patent including Dietzia maris. 
Microorganisms which specifically produce acrylonitrile hydratase enzymes or other analogous enzymes for converting unsaturated nitriles to the corresponding amides or carboxylic acids, are grown at about neutral pH, that is approximately pH 6 to 8. Consequently, it can be more difficult to maintain the sterility during the culturing of the bacterium as it is recognized that many, many microorganisms will grow at this pH. A particular problem that can occur therefore, is that the fermentation can become contaminated with other microorganisms. Such contamination not only impairs the production of the required enzyme, but may also result in undesirable by-products when used to convert the unsaturated nitrile to the desired product. Additionally, it is most undesirable to have other microorganisms present since in order to ensure they are not harmful, that is not pathogenic, the unknown contaminants would have to be identified. Consequently, the fermentation would have to be abandoned which is both expensive and time-consuming.
It has already been described that urea is often added to the fermentation medium as an inducer of the nitrile hydratase of many organisms that are shown to produce acrylamide from acrylonitrile. Solutions of urea can be alkaline due to the presence of ammonium ion in the solution. And additionally if the urea is degraded at all during the fermentation this releases ammonium ion causing the pH of the medium to increase, unless buffering capacity in the form of buffer salts is added at high levels, or more likely the increasing pH effects are counteracted by the use of acid addition to the fermentation. This is therefore a further problem with fermenting the microorganism at neutral pH in that it is normally required to buffer the reaction mixture in order to counteract the effect of adding urea. Buffer solutions that are used may include phosphate salts, citric acid in combination with a basic salt such as phosphate, tris or any other buffer generally known to be applicable to use in fermentation systems to give rise to a neutral pH. Acids that may be used for buffering purposes include phosphoric, acetic, sulphuric and any other that may be suitable for this purpose.
Nitrile
A further problem is that many microorganisms tend not to be tolerant to high salt concentrations and this can result in cell leakage during growth and during use of the bacteria as a biocatalyst due to the differences in the osmotic pressure within the cell and in the fermentation or reaction medium. For instance if a microorganism is being used to prepare a carboxylic acid salt, this solution would have a higher ionic strength than water or buffer solution. It might be the case that depending upon the microorganism used, the difference in the osmotic pressure in the cell and the reaction solution would cause the cell to rupture thus reducing the capability of the organism to act as an effective biocatalyst and also by virtue of the intracellular material now being present as a contaminant of the reaction mixture, which may be wholly undesirable.